Preparation and application of multifunctional antibacterial gel for promoting wound healing

Abstract

The invention discloses a preparation method and application of multifunctional antibacterial gel, and relates to the technical field of biomedical injectable hydrogel materials with antibacterial activity. The cationic guar gum (CG) is used as a cross-linked network and is loaded with a water-soluble Fe (II)-based metal organic metal cage (Fe-1SO4), and through in-situ chemical dynamic therapy (CDT) and photodynamic therapy (PDT) induced under irradiation of 660 nm laser, rapid healing of surface wounds is promoted through synergistic antibiosis. Fe-1SO4 is a water-soluble metal cage which takes Fe (II) as a metal node and 5, 10, 15, 20-tetra (4-aminophenyl)-21H, 23h-porphyrin (TAPP) as an organic ligand, and can be quickly degraded within 5 days in a biological environment. According to the present invention, with the application of the fenton-like reaction in the preparation of the antibacterial agent, the obvious PDT effect can be provided under the irradiation of 660 nm laser so as to generate a large amount of singlet oxygen (1O2), and the fenton-like reaction can convert H2O2 generated by bacterial infection into toxic hydroxyl radicals (OH) in situ, such that the excellent antibacterial activity can be provided in vivo and in vitro, and the wound healing can be promoted. The invention provides a simple strategy for the design of the rapidly degradable antibacterial hydrogel dressing, and is an important step for the clinical application of biological materials.

Description

Technical Field

[0001] The invention relates to the technical field of antibacterial gel, and in particular to a multifunctional antibacterial gel and a preparation method and application thereof. Background Art

[0002] Infectious diseases caused by pathogenic bacteria pose a major threat to global health, and traditional treatments include antibiotic sprays and surgical operations. Among them, medical materials used during surgery are mostly petroleum plastic products, which are difficult to biodegrade, hinder the rapid healing of postoperative wounds, increase the risk of secondary infection, and endanger life. Therefore, the development of rapidly degradable biomedical materials to treat bacterial infections is urgent. In recent years, various new antibacterial materials have been designed as therapeutic agents and are increasingly used in preclinical treatments through various methods, including photothermal therapy (PTT), chemodynamic therapy (CDT), and photodynamic therapy (PDT). Faced with the serious risk of secondary infection in treatment, two treatment methods are usually used in synergy, using the unique antibacterial mechanism of each treatment method to produce an additive therapeutic effect.

[0003] Metal-organic cages (MOCs) are supramolecular cages self-assembled from metal ions or clusters and organic ligands with specific cavity structures, which can be divided into regular (Platonic) polyhedrons, quasi-regular polyhedrons, and semi-regular polyhedrons. They are widely used in various fields, such as catalysis, biomedical applications, host-guest chemistry, molecular recognition and separation. However, most MOCs are difficult to dissolve in aqueous solutions, which seriously limits their development in biomedical applications. In contrast, water-soluble MOCs have received increasing attention in recent years due to their flexibility, controllability and environmental friendliness.

[0004] Therefore, providing an antibacterial gel that can improve the water solubility of MOCs for antibacterial purposes and improve the self-degradation ability of MOCs in a biological environment is an urgent problem to be solved by the present invention. Summary of the invention

[0005] The present invention provides a multifunctional antibacterial gel and a preparation method and application thereof. The gel can maximize the antibacterial effect by combining CDT and PDT, has significant synergistic antibacterial activity both in vitro and in vivo, and has the characteristics of rapid degradation, injectability, high antibacterial efficiency and significant in vivo wound healing ability.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions: In a first aspect, the present invention provides a composite antibacterial agent, wherein the multifunctional antibacterial gel comprises Fe(II)-coordinated MOC (Fe-1•SO 4 ) and guar gum CG.

[0007] Porphyrin and its derivatives as photosensitizers are prone to severe π-π stacking, leading to fluorescence quenching and 1 O 2 The reduced quantum yield of the generated material greatly limits the application of porphyrin and its derivatives in PDT. Therefore, porphyrin derivatives can be used as ligands to self-assemble with metal ions to form water-soluble MOCs of various shapes, which can effectively avoid the π-π stacking of the porphyrin macrocycle and promote 1 O 2 In addition, Fe(II), as a transition metal ion, can catalyze the formation of H in infected tissues through a Fenton-like reaction. 2 O 2 Produces cytotoxic hydroxyl radicals (•OH).

[0008] The present invention utilizes water-soluble Fe(II)-coordinated MOCs (Fe-1•SO 4 ) embedded in a cationic guar gum (CG) network to construct a rapidly biodegradable antibacterial platform (CG@Fe Gel), which was prepared using a simple one-pot mixing method for the treatment of localized infections. TAPP is a typical photosensitizer that can produce a 660 nm laser-induced 1 O 2 , effectively killing bacteria (>50%). At the same time, CG@Fe gel can be used as a mimic peroxidase-responsive facultative anaerobic microenvironment and convert H 2 O 2 Efficiently converted into highly toxic •OH. CG@Fe gel enhances the antibacterial effect by combining CDT and PDT, and exhibits significant synergistic antibacterial activity (>92%) both in vitro and in vivo. In addition, CG@Fe gel achieves rapid biodegradation within 5 days, effectively avoiding the long-term retention of the material in biological tissues, causing tissue damage and functional disorders. This shows that CG@Fe gel has great application prospects in the clinical treatment of drug-resistant bacterial infections.

[0009] The significant advantages of the present invention are: 1. The multifunctional antibacterial hydrogel of the present invention can be used for synergistic antibacterial treatment with PDT and CDT 2. The multifunctional antibacterial hydrogel prepared by the present invention has the characteristics of rapid degradation, injectability, high antibacterial efficiency and significant wound healing ability in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Provide (a) water-soluble Fe-1•SO 4 of 1 H NMR spectrum (b) Water-soluble Fe-1•SO4 Low-resolution ESI mass spectrometry analysis (c) Schematic diagram of the formation of CG@Fe gel by hydrogen bonding interactions between hydroxyl groups and quaternary ammonium groups (d) CG gel (white), Fe-1•SO 4 Photos of solution (dark red liquid) and CG@Fe gel (dark red) Figure 2 (a) UV-visible absorption spectrum. (b, c) Fluorescence intensity of DMA in different groups with irradiation time. (d, e) Changes of UV-visible absorption of MB in different groups with incubation time. (f) EPR spectrum of CG@Fe gel under different measurement conditions.

[0011] Figure 3 Antibacterial activity of the samples: photos of colonies formed by (a) Escherichia coli and (c) Staphylococcus aureus in the dark and after 660 nm laser irradiation for 5 min (n=3, ***p<0.001) and the corresponding bacterial activity of (b) Escherichia coli and (d) Staphylococcus aureus Figure 4 (a) Schematic diagram of the establishment, drug administration and sacrifice of the Staphylococcus aureus infection wound model. (b) Blank (I), CG gel (II), H 2 O 2 +CG gel (III), CG@Fe gel (IV) and H 2 O 2 +CG@Fe gel (V) was irradiated with 660 nm laser (1.0 W / cm 2 ) Representative photos of wounds infected with Staphylococcus aureus treated under different conditions. (c) Corresponding wound healing traces in each treatment group. (d) Wound healing rate of rats from day 0 to day 10 (***p < 0.001). (e) Photo of bacterial colonies on the infected wound on day 2 and g) corresponding colony data, ***p < 0.001. h) H&E staining micrographs of infected wounds treated with different conditions on day 10. (Scale bar: 500 μm). (h) Representative photos of the rapid degradation of CG@Fe gel at different times in BALB / C female mice (scale bar = 4 mm). DETAILED DESCRIPTION

[0012] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to explain the present application and is not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0013] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the data used in this way can be interchanged where appropriate, so that the implementation of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. Example

[0014] Preparation of CG@Fe gel Weigh 50 mg of TAPP, 25.75 μL of 2-pyridinecarboxaldehyde, 31.95 mg of zinc iron trifluoromethanesulfonate, and 1.5 mL of DMF and place them in a three-necked flask. Degas the nitrogen gas by three times of pumping / circulation, and heat at 70 °C for 16 h. Wash with ether to obtain 70.82 mg of the product A•OTf. Then, dissolve the A•OTf cage in CH 3 CN, add tetrabutylammonium sulfate TBA 2 SO 4 solution, the mixture was centrifuged and washed with CH 3 CN:H 2 O(9:1) to thoroughly wash the precipitate. 4 Dark red solid.

[0015] Weigh 4.5 mg Fe-1•SO 4 Dissolved in 2 ml PBS, 10 mg guar gum CG was then slowly added to the Fe-1•SO 4 The solution was stirred continuously until the guar gum CG was evenly dispersed in the solution. Example

[0016] Preparation of CG@Fe gel Weigh 50 mg of TAPP, 25.75 μL of 2-pyridinecarboxaldehyde, 31.95 mg of zinc iron trifluoromethanesulfonate, and 1.5 mL of DMF and place them in a three-necked flask. Degas the nitrogen gas by three times of pumping / circulation, and heat at 70 °C for 16 h. Wash with ether to obtain 70.82 mg of the product A•OTf. Then, dissolve the A•OTf cage in CH 3 CN, add tetrabutylammonium sulfate TBA 2 SO 4solution, the mixture was centrifuged and washed with CH 3 CN:H 2 O(9:1) to thoroughly wash the precipitate. 4 Dark red solid.

[0017] Weigh 4.5 mg Fe-1•SO 4 Dissolved in 2 ml PBS, 15 mg guar gum CG was then slowly added to the Fe-1•SO 4 The solution was stirred continuously until the guar gum CG was evenly dispersed in the solution. Example

[0018] Preparation of CG@Fe gel Weigh 50 mg of TAPP, 25.75 μL of 2-pyridinecarboxaldehyde, 31.95 mg of zinc iron trifluoromethanesulfonate, and 1.5 mL of DMF and place them in a three-necked flask. Degas the nitrogen gas by three times of pumping / circulation, and heat at 70 °C for 16 h. Wash with ether to obtain 70.82 mg of the product A•OTf. Then, dissolve the A•OTf cage in CH 3 CN, add tetrabutylammonium sulfate TBA 2 SO 4 solution, the mixture was centrifuged and washed with CH 3 CN:H 2 O (9:1) to thoroughly wash the precipitate. 4 Dark red solid. Weigh 4.5 mg Fe-1•SO 4 Dissolved in 2 ml PBS, 20 mg guar gum CG was then slowly added to the Fe-1•SO 4 The solution was stirred continuously until the guar gum CG was evenly dispersed in the solution.

[0019] Experimental Section 1. Materials Guar gum (CG) and TAPP were provided by Bidex Pharmaceuticals (Shanghai, China). Hydrogen peroxide (30 wt %), anhydrous ethanol, and 9,10-dimethylanthracene (DMA) were purchased from Aladdin (Shanghai, China). Live / dead™ BacLight™ Bacterial Viability Assay Kit, consisting of propidium iodide (PI) and SYTO9, was purchased from Thermo Fisher Scientific (L7012, Waltham, USA). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin, and phosphate-buffered saline (PBS, pH=7.4) were purchased from Beyotime Biotechnology (Shanghai, China). All reagents were used without further purification or modification.

[0020] 2. Instruments 1 H NMR spectra were recorded by an Agilent 600 MHz DD2 spectrometer. Bacterial solutions were obtained by a constant temperature incubator (MJ-150-I, China). The morphologies of different samples were observed by scanning electron microscopy (SEM, FEI NOVA 400). The UV-visible absorption spectra of the samples were measured by SP-756P UV-visible spectrometer. A 660 nm laser was prepared using a DS660-MFC-2 laser light source. The chemical composition of the samples was analyzed by a Fourier transform infrared spectrometer (FLIR A35 FOV 25 (60 Hz)). Electron paramagnetic resonance (EPR) measurements were performed on a Bruker A300 instrument. The rheological properties of the hydrogels were determined by a rheometer (Discovery HR10, TA, USA).

[0021] 3. In vitro photodynamic and chemokinetic properties of CG@Fe gel Detection using a specific quencher, 9,10-dimethylanthracene (DMA) 1 O 2 DMA methanol solution (50 µL, 0.1 mg) was added to 4 mL of a mixed solvent of ethanol and water (4 / 1,). The fluorescence spectrum of DMA was detected every 1 min. The fluorescence spectrum of the CG@Fe Gel illumination group decreased significantly compared with other groups, indicating that CG@Fe Gel has the ability to produce PDT. 1 O 2 In addition, since the Fe ions in CG@Fe gel can catalyze the Fenton-like reaction to generate hydroxyl radicals (•OH) with antibacterial activity, we used methylene blue (MB) as an indicator to evaluate the catalytic activity of CG@Fe gel in H 2 O 2The ability to generate •OH free radicals. In the CG@Fe gel group, the absorption peak of MB at 665 nm decreased over time, and the degradation of MB once again confirmed that CG@Fe gel has the ability to generate •OH in a time-dependent manner. In addition, the types of ROS generated by CG@Fe gel were studied by EPR. 2 O 2 In the +CG@Fe Gel group, it was observed 1 O 2 1:1:1 electron cyclotron resonance of free radicals and 1:2:2:1 electron cyclotron resonance of •OH radicals.

[0022] 4. In vitro antibacterial test Different concentrations of Fe-1•SO 4 The photodynamic antibacterial effect was evaluated under light / dark conditions. The CG@Fe gel had no antibacterial activity under dark conditions. 4 When the concentration increased to 100 μM, CG@Fe gel was exposed to 660 laser (1.0 W / cm 2 ) showed obvious antibacterial effect after irradiation for 10 min. 4 When the concentration was 100 μM, the chemical kinetic antibacterial effect of CG@Fe gel increased with the increase of H 2 O 2 concentration. 2 O 2 When the concentration increased to 2.0 mM, it had an excellent antibacterial effect within 10 min, which was due to the antibacterial effect of CG@Fe gel generating •OH through Fenton-like reaction. Therefore, chemical dynamic antibacterial can complement the photodynamic antibacterial effect to maximize the antibacterial effect of CG@Fe gel and achieve the purpose of synergistically killing pathogenic bacteria in wound tissue. 2 O 2 After treatment with +CG gel alone, the number of Escherichia coli and Staphylococcus aureus colonies was relatively large. 2 O 2 +CG@Fe Gel dark group showed certain PDT and CDT activity against Escherichia coli and Staphylococcus aureus within 5 min, respectively. Excitingly, under 660 nm laser (1.0 W / cm 2 ) with the help of H 2 O 2 +CG@Fe Gel illumination group inactivated almost all Escherichia coli (92.59%) and Staphylococcus aureus (95.86%) within 5 minutes.

[0023] 5. Mechanical properties of multifunctional antibacterial gel As a wound dressing, the multifunctional antibacterial gel needs to have specific mechanical properties to meet the needs of different irregular and deep wounds. First, the rheological sweep results show that in the gradually increasing frequency range of 0.1 to 10 Hz, G' is always greater than G", indicating that the elastic behavior of CG@Fe gel is dominant. When the shear rate increases from 1 s -1 Increase to 100 s -1 The viscosity of CG@Fe gel decreased when the CG@Fe gel was physically cut into two halves. After 3 minutes, the two cut parts automatically reconnected without any external intervention and reassembled into a new cut part without obvious fracture lines, which verified the self-healing ability of the hydrogel. 4 The CG@Fe gel was immersed in PBS solution containing 1 mg / mL lysozyme at 37 °C for 5 days. 4 Both CG@Fe and CG@Fe Gel can be rapidly degraded to evaluate their biodegradability. This ensures that they will not cause tissue damage or functional impairment when used as wound dressings. Subsequently, we studied the dynamic swelling ratio and water retention capacity of CG@Fe gel, and the results showed that the designed CG@Fe gel has excellent water retention capacity and can maintain a moist environment that is conducive to wound repair for a long time.

[0024] 6. PDT / CDT in vivo antibacterial test A circular wound with a diameter of 1 cm was cut on the back of Sprague-Dawley rats, and Staphylococcus aureus solution (100 μL, 1.0x108 CFU / mL) was dripped into the wound to establish a wound infection model. Staphylococcus aureus is the most common microorganism in wound infection. After injection into the wound on the back of the rat skin, the wound was photographed and the wound size was processed using Image J software. Subsequently, the skin tissue was homogenized in sterile PBS and cultured on an agar plate overnight. 2 O 2 There were a large number of colonies on the agar plate of the +CG gel group, and the number of bacteria in the CG@Fe Gel light-treated group began to decrease. 2 ) 5 min later, H 2 O 2 There were almost no bacteria in the +CG@Fe gel group, which was consistent with the results of in vitro experiments.

[0025] In addition, to verify the above healing results, we evaluated the details of wound healing by histological changes in skin tissue. On the 10th day of treatment, H&E staining was used to evaluate the infiltration of inflammatory cells in the wound tissue.2 O 2 +CG@Fe Gel In the light-exposed group, there was a small amount of inflammatory cells, mainly neutrophil infiltration. In addition, no significant changes were observed in the body weight of all rats. These results further indicate that the designed CG@Fe gel antibacterial dressing has almost no cytotoxicity. As we know, polysaccharide hydrogels are biodegradable materials. The rapid biodegradation effect of CG@Fe gel in vivo indicates that CG@Fe gel has great clinical application potential as a wound dressing. The volume was significantly reduced 2 days after injection, and the dissolution results in about 5 days indicated that CG@Fe gel can be used as a multifunctional antibacterial gel for PDT / CDT synergistic treatment.

[0026] 7. Statistical analysis The collected quantitative data are expressed as mean ± SD. Statistical significance was analyzed by Student's t-test. Statistical significance was inferred as ***p < 0.001.

[0027] In summary, the present invention constructs a Fe(II)-coordinated water-soluble Fe-1•SO 4 The PDT and CDT synergistic multifunctional antibacterial hydrogel (CG@Fe Gel) composed of Fe-1•SO and cationic guar gum (CG) has fast degradation, injectability, high antibacterial efficiency and significant in vivo wound healing ability. 4 The TAPP ligand was used as a photosensitizer to generate a photoreceptor with the ability to damage cancer cells under 660 nm laser irradiation through PDT. 1 O 2 In addition, Fe-1•SO 4 The Fe ions in the bacteria can respond to the bacterial microenvironment and convert H 2 O 2 Efficiently converted into highly toxic •OH. Therefore, by combining CDT and PDT, CG@Fe gel can maximize its antibacterial effect and has significant synergistic antibacterial activity (>92%) both in vitro and in vivo. It is worth noting that CG@Fe gel rapidly degrades within 5 days in a biological environment, effectively avoiding the accumulation of biomaterials and causing damage to normal biological tissue function. In general, the present invention proposes a multifunctional synergistic antibacterial platform that combines PDT and CDT to achieve efficient antibacterial treatment. This new platform has taken an important step towards promoting the clinical application of biodegradable antibacterial materials. The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A multifunctional antibacterial gel, characterized in that: The Fe(II)-coordinated water-soluble MOCs (Fe-1•SO4) were embedded in a cationic guar gum (CG) network, and the mixture rapidly formed a hydrogel in a Fe-1•SO4 solution due to the hydrogen bonding interactions between the hydrophilic polysaccharides of the cationic CG network.

2. The multifunctional antibacterial gel according to claim 1, characterized in that: In the multifunctional antibacterial gel, Fe(II)-coordinated MOCs (Fe-1•SO4) has good water solubility and has a synergistic therapeutic effect of PDT and CDT.

3. The composite antibacterial agent according to claim 2, characterized in that: In the antimicrobial agent, guar gum rapidly combines with Fe(II)-coordinated MOCs (Fe-1•SO4).

4. The method for preparing the multifunctional antibacterial gel according to any one of claims 1 to 3, characterized in that: In the multifunctional antibacterial gel, Fe(II)-coordinated MOCs (Fe-1•SO4) and guar gum CG are obtained by a simple one-pot method.

5. Use of the multifunctional antibacterial gel as claimed in claim 4 in preparing an efficient and degradable wound antibacterial gel material.

6. The use according to claim 5, characterized in that: The bacteria are Escherichia coli or Staphylococcus aureus.

7. The method for controlling bacteria on a wound of a mammal using the multifunctional antibacterial gel according to any one of claims 1 to 3, characterized in that: An effective dose of multifunctional antibacterial gel is applied to the wound of the mammal.

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